The invention relates to anti-freeze polypeptides (AFPs) and food product containing AFPs.
Anti-freeze polypeptides (AFPs) have been suggested for improving the freezing tolerance of foodstuffs.
For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which inhibit the growth of ice-crystals. See for example U.S. Pat. No. 5,111,792.
WO 90/13571 discloses, antifreeze peptides produced chemically or by recombinant DNA techniques from slants. The AFPs can suitably be used in food-products such as ice-cream. Example 3B shows modified ice-crystal shapes if a water-ice mixture is frozen into a film in combination with 0.01 wt % of AFP.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from intercellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plant cells.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Up till now, however the use of AFPs has not been applied to commercially available consumer products. One reason for this are the high costs and complicated process for obtaining AFPs. Another problem is that sources of the AFPs are either difficult to obtain in sufficient quantities (e.g. fish containing AFPs) or are not directly suitable for use in food products.
The present invention aims to provide novel antifreeze polypeptides which have the advantage that they can easily be obtained from an abundant natural source and which provide good properties to products in which they are used.
It has been found that antifreeze polypeptides which possess good recrystallisation inhibition properties can be obtained from carrots. In particular it has been found that antifreeze polypeptides obtained from carrots show markedly better properties as compared to polypeptides isolated from other root vegetables. In particular the antifreeze polypeptides of the invention are capable of providing good recrystallisation inhibition properties without significantly changing the crystal shape of the ice-crystals, therewith possible leading to more favourable properties e.g. soft ice-cream.
Applicants have found that the effective antifreeze polypeptides from carrots are generally characterised by an apparent Molecular Weight on SDS-PAGE of 36 kDa. Accordingly in a first aspect the invention relates to antifreeze polypeptides which can be obtained from carrots and which have an apparent molecular weight on SDS-PAGE of 36 kDa.
In this context it will be clear to the skilled person that due to variation e.g. in SDS PAGE, the apparent molecular weight can only be determined with some variation in the results. For the purpose of the invention these variations e.g. from 30 to 40 kDa or from 34 to 39 kDa are also embraced within the scope of the term xe2x80x9capparent Molecular Weight of 36 kDaxe2x80x9d.
Applicants also have found that the effective anti-freeze polypeptides according to the invention comprise fragments having an amino acid sequence as represented in the examples.
Accordingly in a second aspect the invention relates to polypeptides comprising one or more fragments (A-E) having an amino add sequence as follows:
SEQ ID NOS. 1-5, respectively, in order of appearance.
(A) LEU-PRC-ASN-LEU-PHE-GLY-LYS
(B) ILE-PRO-GLU-GLU-ILE-SER-ALA-LEU-LYS
(C) LEU-THR-X-LEU-ASP-LEU-SER-PHE-ASN-LYS
(D) SER-LEU-ARG-LEU-SER-SER-THR-SER-LEU-SER-GLY-PRO-VAL-PRO-LEU-PHE-PHE-PRO-GLN-LEU-X-LYS
(E) X-X-GLU-VAL-ILE-PRO-X-GLN-LEU-SER-THR-LEU-PRO-ASN-LEU-LYS
Preferably the AFPs of the invention comprise all of the partial sequences (A-E).
The complete amino acid sequence of the preferred AFP of the invention is represented below. Accordingly, in a third aspect the invention relates to an anti-freeze protein having an amino acid sequence as shown in Listing 1:
Also embraced within the invention are isoforms and derivatives of the above mentioned polypeptides which still possess the antifreeze properties. Preferable the derivatives show at least 75% homology with the polypeptide of Listing 1 or the polypeptide comprising the partial sequences (A-E) more preferred more than 85%, most preferred more than 95%. For the purpose of the invention the term derivative also embraces modified polypeptides which still possess the antifreeze properties, for example glycosylated forms of the above polypeptides.
Also embraced within the invention are nucleotide sequences encoding the amino acids as described above. In particular the invention relates to nucleotide sequences of Listing 1 and alleles thereof.
Also embraced within the invention are nucleotide fragments derived from the coding region that are capable of hybridizing to related genes that code for anti-freeze peptides.
Although the proteins of the invention can easily directly be isolated from carrots, also genetic manipulation techniques may be used to produce the proteins described in the invention.
An appropriate host cell or organism would be transformed by a gene construct that encodes the desired polypeptide. The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector containing the necessary elements for transcription and translation and in a manner that they will be expressed under appropriate conditions (eg in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.
A number of expression systems may be utilised to express the polypeptide coding sequence. These include, but are not limited to, bacteria, yeast, insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors. Yeast, plants and plant culture systems are preferred in this context.
A wide variety of plants and plant cell systems can be transformed with the nucleic acid constructs of the polypeptides. Preferred embodiments would include, but are not limited to, maize, tomato, tobacco, carrots, strawberries, rape seed and sugar beet.
One preferred embodiment of the invention relates to the use of AFPs of the invention for increasing the frost tolerance of plants. This case for example be done by the above method whereby the plants are transformed to ensure. (increased) production of the AFPs of the invention, therewith increasing the frost tolerance of said plants.
The invention also relates to antibodies which specifically bind an (epitope of the) polypeptides of the invention. Also embraced are polypeptides which are immunologically related to the polypeptides as determined by its cross reactivity with an antibody raised against the above polypeptides.
Based on the above information it is also possible to genetically modify other natural sources such that they produce the advantageous AFPs as identified here-above.
Preferably those AFPs are chosen which have significant ice-recrystallisation inhibition properties. A suitable test for determining the recrystallisation inhibition properties is indicated in example I. Also preferably AFPs in accordance to the invention provide a ice particle size in the frozen product(mean crystal length) upon recrystallisation of less than 50 xcexcM, more preferred from 5 to 40 xcexcm.
The AFPs can conveniently be used in several products, preferably in food products which are frozen or intended to be frozen. Carrots which comprise the AFP at naturally occuring levels are not embraced within the scope of the invention. However, food product containing (parts) of carrots are embraced within this term. Also embraced are carrots which have been transformed to over express the AFP of the invention i.e. which contain the AFP at significantly higher levels than non-transformed carrots.
Examples of such food products are: frozen food products such as vegetables, sauces, soups, snacks, frozen confectionery products such as ice-cream or water-ice, dairy products etc.
The preferred products wherein the AFPs are used are or frozen vegetables or frozen confectionery products such as ice-cream or water-ice. Preferably the level of AFPs is from 0.00001 to 0.5 wt % based on the final product.
If dry-mixes or concentrates are used, the concentration may be higher in order to ensure that the level in the final frozen product is within the above ranges. Surprisingly it has been found that compositions of the invention can contain very low amounts of AFPs while still being of good quality.
Preferred levels of AFP are from 0.00001 to 0.5 wt %, more preferred 0.00005 to 0.3 wt %, most preferred 0.0001 to 0.2 wt %.
For the purpose of the invention it is not necessary to add the AFP in purified form to the food product. Also possible is to add a composition comprising AFPs e.g. an extract of the natural material which produces the AFP.
Also it is possible to modify the food product such that the AFP is produced in situ e.g. by adding genetically modified micro-organisms which are capable of producing the AFP in the food product, or even to genetically modify the food product (e.g. the vegetable) such that (the vegetable) in itself it is capable of producing the AFP in situ.
For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granites and frozen fruit purees.
Preferably a the level of solids in the frozen confection (e.g. sugar, fat, flavouring etc) is more than 3 wt %, more preferred from 10 to 70 wt, for example 40 to 70 wt %.
Frozen confectionery products according to the invention can be produced by any method suitable for the production of frozen confectionery. Especially preferably however all the ingredients of the formulation are fully mixed before the freezing process starts.